It has been estimated that about 5-10% of breast cancer is inherited Rowell, et al., American Journal of Human Genetics, 55:861-865 (1994). Located on chromosome 17, BRCA1 is the first gene identified conferring increased risk for breast and ovarian cancer. Miki et al., Science, 266:66-71 (1994). Mutations in this tumor suppressor gene account for roughly 45% of inherited breast cancer and 80-90% of families with increased risk of early onset breast and ovarian cancer. Easton et al., American Journal of Human Genetics. 52:678-701 (1993).
The location of one or more mutations in the BRCA1 region of chromosome 17 provides a promising approach to reducing the high incidence and mortality associated with breast and ovarian cancer through the early detection of women at high risk. These women, once identified, can be targeted for more aggressive prevention programs. These mutations are also associated with an increased risk of ovarian cancer, prostrate cancer and pancreatic cancer. Screening is carried out by a variety of methods which include karyotyping, probe binding and DNA sequencing.
In DNA sequencing technology, genomic DNA is extracted from whole blood and the coding regions of the BRCA1 gene are amplified using the polymerase chain reaction (PCR). Each of the coding regions is sequenced completely and the results are compared to the normal DNA sequence of the gene (GENBANK Accession Numbers: U14680 and U15595). Many mutations and several polymorphisms have already been reported in the BRCA1 gene. Shattuck-Eidens, et al., Journal of the American Medical Association, 273: 535-541 (1995).
The BRCA1 gene (GENBANK Accession Numbers: U14680 and 15595) is divided into 24 separate exons. Exons 1 and 4 are noncoding, in that they are not part of the final functional BRCA1 protein product. The BRCA1 coding region spans roughly 5600 base pairs (bp). Each exon consists of 200-400 bp, except for exon 11 which contains about 3600 bp. To sequence the coding region of the BRCA1 gene, each exon is amplified separately and the resulting PCR products are sequenced in the forward and reverse directions. Because exon 11 is so large, it was divided it into multiple overlapping PCR fragments.
There is a need in the art to identify mutations in the BRCA1 gene. Identification of mutations of the BRCA1 gene and protein would allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible.
Many mutations and normal polymorphisms have already been reported in the BRCA1 gene. A world wide web site has been built to facilitate the detection and characterization of alterations in breast cancer susceptibility genes. Such mutations in BRCA1 can be accessed through the Breast Cancer Information Core at: HTTP://www.nchgr.nih.gov/dir/lab.sub.-- transfer/bic.
While mutations occur throughtout the BRCA1 gene, there is a need for a high sample number (throughput), sensitivity, accuracy and cost effectiveness. Identification of mutations of the BRCA1 gene would allow more widespread diagnostic screening for hereditary breast and ovarian cancer than is currently possible and permit identification of functional areas deduced from the mutational spectrum observed.